Tuberculosis (TB) is a major cause of human mortality with 9 million new cases and nearly two million deaths annually; approximately two billion people are infected with the causative agent, Mycobacterium tuberculosis. In Argentina there are about 12,000 cases and one thousand deaths per year. While M. tuberculosis infections can be effectively resolved with a standard 6-9 month course of antibiotics with at least three drugs, the emergence of drug resistant strains severely complicates treatment. There is a need for new diagnostic approaches that combine speed (time-to-detection), sensitivity, specificity, biosafety, rapid and accurate determination of resistance to the commonly used anti-tuberculosis drugs at a low cost to be applied in developing countries where the incidence of TB is high. Mycobacteriophages are excellent candidates for the development of diagnostic tools since they efficiently and specifically infect and replicate in Mycobacteria. We recently described the development of Fluoromycobacteriophages - reporter phages containing a fluorescent reporter gene - that provide a simple means of revealing the metabolic state of M. tuberculosis cells, and therefore their response to antibiotics. Fluorescence can be detected easily by fluorescent microscopy or by flow cytometry. The assay is responsive to antibiotics, and fluorescence is maintained for at least two weeks following fixation, increasing biosafety and facilitating storage or transportation of samples. Fluoromycobacteriophages have promising attributes in the research laboratory, and our goal is to develop the next generation of fluoromycobacteriophages that can be used for direct analysis of clinical samples, with a readout within one hour. We propose to modify the current phages such DNA replication contributes to signal amplification, which can be accomplished by the construction of lysis- defective phage mutants; this is a particularly desirable feature for use in developing countries, since they can be used at any infection temperature. Incorporation of optimized versions of fluorescent genes with an enhanced mycobacterial expression will also enhance the signal and shorten the time-to-detection. We also propose to develop a system for addition of affinity tags to phage particles to ensure efficient capture of mycobacterial cells, and thus optimize the sensitivity of detection. Finally, the construction of these optimized versions of Fluoromycobacteriophages will facilitate the testing of specific protocols for sputum processing to achieve efficient phage infection of mycobacterial cells directly in these samples. Together, these developments will result in a simple, rapid, and specific diagnostic test for tuberculosis. This research will be primarily done in Argentina at University of Buenos Aires in collaboration with Dr. Mariana Piuri as an extension of NIH Grant AI064494 (7/1/06 - 6/30/11).